LOCUS Exported 7961 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION synthetic circular DNA ACCESSION . VERSION . KEYWORDS pTALEN_v2 (NI) SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 7961) AUTHORS Sanjana NE, Cong L, Zhou Y, Cunniff MM, Feng G, Zhang F TITLE A transcription activator-like effector toolbox for genome engineering. JOURNAL Nat Protoc. 2012 Jan 5;7(1):171-92. doi: 10.1038/nprot.2011.431. PUBMED 22222791 REFERENCE 2 (bases 1 to 7961) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..7961 /organism="synthetic DNA construct" /mol_type="other DNA" rep_origin 86..674 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 575..594 /label=pBR322ori-F /note="pBR322 origin, forward primer" primer_bind 828..845 /label=L4440 /note="L4440 vector, forward primer" primer_bind 943..960 /label=M13 Forward /note="In lacZ gene. Also called M13-F20 or M13 (-21) Forward" primer_bind 944..960 /label=M13 fwd /note="common sequencing primer, one of multiple similar variants" protein_bind 1416..1432 /label=lac operator /bound_moiety="lac repressor encoded by lacI" /note="The lac repressor binds to the lac operator to inhibit transcription in E. coli. This inhibition can be relieved by adding lactose or isopropyl-beta-D-thiogalactopyranoside (IPTG)." enhancer 1459..1838 /label=CMV enhancer /note="human cytomegalovirus immediate early enhancer" promoter 1839..2042 /label=CMV promoter /note="human cytomegalovirus (CMV) immediate early promoter" primer_bind 1993..2013 /label=CMV-F /note="Human CMV immediate early promoter, forward primer" primer_bind 2087..2106 /label=T7 /note="T7 promoter, forward primer" promoter 2087..2105 /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" regulatory 2107..2116 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" CDS 2116..2181 /codon_start=1 /product="three tandem FLAG(R) epitope tags, followed by an enterokinase cleavage site" /label=3xFLAG /translation="DYKDHDGDYKDHDIDYKDDDDK" CDS 2188..2208 /codon_start=1 /product="nuclear localization signal of SV40 (simian virus 40) large T antigen" /label=SV40 NLS /translation="PKKKRKV" promoter 2677..2707 /label=lac UV5 promoter /note="E. coli lac promoter with an ""up"" mutation" CDS 2761..3420 /codon_start=1 /gene="cat" /product="chloramphenicol acetyltransferase" /label=CmR /note="confers resistance to chloramphenicol" /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA" primer_bind complement(2827..2846) /label=CAT-R /note="Chloramphenicol resistance gene, reverse primer" CDS 3762..4067 /codon_start=1 /gene="ccdB" /product="CcdB, a bacterial toxin that poisons DNA gyrase" /label=ccdB /note="Plasmids containing the ccdB gene cannot be propagated in standard E. coli strains." /translation="MQFKVYTYKRESRYRLFVDVQSDIIDTPGRRMVIPLASARLLSDK VSRELYPVVHIGDESWRMMTTDMASVPVSVIGEEVADLSHRENDIKNAINLMFWGI" primer_bind 3946..3965 /label=ccdB-fwd /note="ccdB gene, forward primer" CDS 4371..4958 /codon_start=1 /product="nonspecific DNA cleavage domain of the FokI endonuclease (Li et al., 1992)" /label=FokI cleavage domain /note="must dimerize to be catalytically active" /translation="QLVKSELEEKKSELRHKLKYVPHEYIELIEIARNSTQDRILEMKV MEFFMKVYGYRGKHLGGSRKPDGAIYTVGSPIDYGVIVDTKAYSGGYNLPIGQADEMQR YVEENQTRNKHINPNEWWKVYPSSVTEFKFLFVSGHFKGNYKAQLTRLNHITNCNGAVL SVEELLIGGEMIKAGTLTLEEVRRKFNNGEINF" primer_bind complement(4976..4993) /label=BGH-rev /note="Bovine growth hormone terminator, reverse primer. Also called BGH reverse" polyA_signal 4982..5093 /label=bGH poly(A) signal /note="bovine growth hormone polyadenylation signal" primer_bind complement(5211..5231) /label=pBABE 3' /note="SV40 enhancer, reverse primer for pBABE vectors" promoter 5216..5545 /label=SV40 promoter /note="SV40 enhancer and early promoter" rep_origin 5396..5531 /label=SV40 ori /note="SV40 origin of replication" primer_bind 5458..5477 /label=SV40pro-F /note="SV40 promoter/origin, forward primer" CDS 5594..6619 /codon_start=1 /gene="aph(4)-Ia" /product="aminoglycoside phosphotransferase from E. coli" /label=HygR /note="confers resistance to hygromycin" /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK E" polyA_signal 6749..6870 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(6786..6805) /label=SV40pA-R /note="SV40 polyA, reverse primer" primer_bind 6840..6859 /label=EBV-rev /note="SV40 polyA terminator, reverse primer" CDS complement(6923..7783) /codon_start=1 /gene="bla" /product="beta-lactamase" /label=AmpR /note="confers resistance to ampicillin, carbenicillin, and related antibiotics" /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS LIKHW" primer_bind 7546..7565 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(7784..7875) /gene="bla" /label=AmpR promoter primer_bind 7917..7936 /label=Kan-R /note="Kanamycin resistance gene, reverse primer" primer_bind 7942..7961 /label=pENTR-R /note="pENTR vectors, reverse primer" ORIGIN 1 ctcatgacca aaatccctta acgtgagtta cgcgcgcgtc gttccactga gcgtcagacc 61 ccgtagaaaa gatcaaagga tcttcttgag atcctttttt tctgcgcgta atctgctgct 121 tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa gagctaccaa 181 ctctttttcc gaaggtaact ggcttcagca gagcgcagat accaaatact gttcttctag 241 tgtagccgta gttagcccac cacttcaaga actctgtagc accgcctaca tacctcgctc 301 tgctaatcct gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt accgggttgg 361 actcaagacg atagttaccg gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca 421 cacagcccag cttggagcga acgacctaca ccgaactgag atacctacag cgtgagctat 481 gagaaagcgc cacgcttccc gaagggagaa aggcggacag gtatccggta agcggcaggg 541 tcggaacagg agagcgcacg agggagcttc cagggggaaa cgcctggtat ctttatagtc 601 ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt gtgatgctcg tcaggggggc 661 ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc ttttgctggc 721 cttttgctca catgttcttt cctgcgttat cccctgattc tgtggataac cgtattaccg 781 cctttgagtg agctgatacc gctcgccgca gccgaacgac cgagcgcagc gagtcagtga 841 gcgaggaagc ggaaggcgag agtagggaac tgccaggcat caaactaagc agaaggcccc 901 tgacggatgg cctttttgcg tttctacaaa ctctttctgt gttgtaaaac gacggccagt 961 cttaagctcg ggccccctgg gcggttctga taacgagtaa tcgttaatcc gcaaataacg 1021 taaaaacccg cttcggcggg tttttttatg gggggagttt agggaaagag catttgtcag 1081 aatatttaag ggcgcctgtc actttgcttg atatatgaga attatttaac cttataaatg 1141 agaaaaaagc aacgcacttt aaataagata cgttgctttt tcgattgatg aacacctata 1201 attaaactat tcatctatta tttatgattt tttgtatata caatatttct agtttgttaa 1261 agagaattaa gaaaataaat ctcgaaaata ataaagggaa aatcagtttt tgatatcaaa 1321 attatacatg tcaacgataa tacaaaatat aatacaaact ataagatgtt atcagtattt 1381 attatcattt agaataaatt ttgtgtcgcc cttaattgtg agcggataac aattacgagc 1441 ttcatgcaca gtggcgttga cattgattat tgactagtta ttaatagtaa tcaattacgg 1501 ggtcattagt tcatagccca tatatggagt tccgcgttac ataacttacg gtaaatggcc 1561 cgcctggctg accgcccaac gacccccgcc cattgacgtc aataatgacg tatgttccca 1621 tagtaacgcc aatagggact ttccattgac gtcaatgggt ggagtattta cggtaaactg 1681 cccacttggc agtacatcaa gtgtatcata tgccaagtac gccccctatt gacgtcaatg 1741 acggtaaatg gcccgcctgg cattatgccc agtacatgac cttatgggac tttcctactt 1801 ggcagtacat ctacgtatta gtcatcgcta ttaccatggt gatgcggttt tggcagtaca 1861 tcaatgggcg tggatagcgg tttgactcac ggggatttcc aagtctccac cccattgacg 1921 tcaatgggag tttgttttgg caccaaaatc aacgggactt tccaaaatgt cgtaacaact 1981 ccgccccatt gacgcaaatg ggcggtaggc gtgtacggtg ggaggtctat ataagcagag 2041 ctctctggct aactagagaa cccactgctt actggcttat cgaaattaat acgactcact 2101 ataggggcca ccatggacta taaggaccac gacggagact acaaggatca tgatattgat 2161 tacaaagacg atgacgataa gatggcccca aagaagaagc ggaaggtcgg tatccacgga 2221 gtcccagcag ccgtagattt gagaactttg ggatattcac agcagcagca ggaaaagatc 2281 aagcccaaag tgaggtcgac agtcgcgcag catcacgaag cgctggtggg tcatgggttt 2341 acacatgccc acatcgtagc cttgtcgcag caccctgcag cccttggcac ggtcgccgtc 2401 aagtaccagg acatgattgc ggcgttgccg gaagccacac atgaggcgat cgtcggtgtg 2461 gggaaacagt ggagcggagc ccgagcgctt gaggccctgt tgacggtcgc gggagagctg 2521 agagggcctc cccttcagct ggacacgggc cagttgctga agatcgcgaa gcggggagga 2581 gtcacggcgg tcgaggcggt gcacgcgtgg cgcaatgcgc tcacgggagc acccctcaac 2641 ctgacagaga ccgcggccgc attaggcacc ccaggcttta cactttatgc ttccggctcg 2701 tataatgtgt ggattttgag ttaggatccg tcgagatttt caggagctaa ggaagctaaa 2761 atggagaaaa aaatcactgg atataccacc gttgatatat cccaatggca tcgtaaagaa 2821 cattttgagg catttcagtc agttgctcaa tgtacctata accagaccgt tcagctggat 2881 attacggcct ttttaaagac cgtaaagaaa aataagcaca agttttatcc ggcctttatt 2941 cacattcttg cccgcctgat gaatgctcat ccggaattcc gtatggcaat gaaagacggt 3001 gagctggtga tatgggatag tgttcaccct tgttacaccg ttttccatga gcaaactgaa 3061 acgttttcat cgctctggag tgaataccac gacgatttcc ggcagtttct acacatatat 3121 tcgcaagatg tggcgtgtta cggtgaaaac ctggcctatt tccctaaagg gtttattgag 3181 aatatgtttt tcgtctcagc caatccctgg gtgagtttca ccagttttga tttaaacgtg 3241 gccaatatgg acaacttctt cgcccccgtt ttcaccatgg gcaaatatta tacgcaaggc 3301 gacaaggtgc tgatgccgct ggcgattcag gttcatcatg ccgtttgtga tggcttccat 3361 gtcggcagaa tgcttaatga attacaacag tactgcgatg agtggcaggg cggggcgtaa 3421 agatctggat ccggcttact aaaagccaga taacagtatg cgtatttgcg cgctgatttt 3481 tgcggtataa gaatatatac tgatatgtat acccgaagta tgtcaaaaag aggtatgcta 3541 tgaagcagcg tattacagtg acagttgaca gcgacagcta tcagttgctc aaggcatata 3601 tgatgtcaat atctccggtc tggtaagcac aaccatgcag aatgaagccc gtcgtctgcg 3661 tgccgaacgc tggaaagcgg aaaatcagga agggatggct gaggtcgccc ggtttattga 3721 aatgaacggc tcttttgctg acgagaacag gggctggtga aatgcagttt aaggtttaca 3781 cctataaaag agagagccgt tatcgtctgt ttgtggatgt acagagtgat attattgaca 3841 cgcccgggcg acggatggtg atccccctgg ccagtgcacg tctgctgtca gataaagtct 3901 cccgtgaact ttacccggtg gtgcatatcg gggatgaaag ctggcgcatg atgaccaccg 3961 atatggccag tgtgccggtc tccgttatcg gggaagaagt ggctgatctc agccaccgcg 4021 aaaatgacat caaaaacgcc attaacctga tgttctgggg aatataaatg tcaggctccc 4081 ttatacacag ccagtctgca ggtcgacggt ctcgactcac gcctgagcag gtagtggcta 4141 ttgcatccaa catcgggggc agacccgcac tggagtcaat cgtggcccag ctttcgaggc 4201 cggaccccgc gctggccgca ctcactaatg atcatcttgt agcgctggcc tgcctcggcg 4261 gacgacccgc cttggatgcg gtgaagaagg ggctcccgca cgcgcctgca ttgattaagc 4321 ggaccaacag aaggattccc gagaggacat cacatcgagt ggcaggttcc caactcgtga 4381 agagtgaact tgaggagaaa aagtcggagc tgcggcacaa attgaaatac gtaccgcatg 4441 aatacatcga acttatcgaa attgctagga actcgactca agacagaatc cttgagatga 4501 aggtaatgga gttctttatg aaggtttatg gataccgagg gaagcatctc ggtggatcac 4561 gaaaacccga cggagcaatc tatacggtgg ggagcccgat tgattacgga gtgatcgtcg 4621 acacgaaagc ctacagcggt gggtacaatc ttcccatcgg gcaggcagat gagatgcaac 4681 gttatgtcga agaaaatcag accaggaaca aacacatcaa tccaaatgag tggtggaaag 4741 tgtatccttc atcagtgacc gagtttaagt ttttgtttgt ctctgggcat ttcaaaggca 4801 actataaggc ccagctcaca cggttgaatc acattacgaa ctgcaatggt gcggttttgt 4861 ccgtagagga actgctcatt ggtggagaaa tgatcaaagc gggaactctg acactggaag 4921 aagtcagacg caagtttaac aatggcgaga tcaatttccg ctcataaaaa atcagcctcg 4981 actgtgcctt ctagttgcca gccatctgtt gtttgcccct cccccgtgcc ttccttgacc 5041 ctggaaggtg ccactcccac tgtcctttcc taataaaatg aggaaattgc atcacaacac 5101 tcaaccctat ctcggtctat tcttttgatt tataagggat tttgccgatt tcggcctatt 5161 ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa ttaattctgt ggaatgtgtg 5221 tcagttaggg tgtggaaagt ccccaggctc cccagcaggc agaagtatgc aaagcatgca 5281 tctcaattag tcagcaacca ggtgtggaaa gtccccaggc tccccagcag gcagaagtat 5341 gcaaagcatg catctcaatt agtcagcaac catagtcccg cccctaactc cgcccatccc 5401 gcccctaact ccgcccagtt ccgcccattc tccgccccat ggctgactaa ttttttttat 5461 ttatgcagag gccgaggccg cctctgcctc tgagctattc cagaagtagt gaggaggctt 5521 ttttggaggc ctaggctttt gcaaaaagct cccgggagct tgtatatcca ttttcggatc 5581 tgatcagcac gtgatgaaaa agcctgaact caccgcgacg tctgtcgaga agtttctgat 5641 cgaaaagttc gacagcgtct ccgacctgat gcagctctcg gagggcgaag aatctcgtgc 5701 tttcagcttc gatgtaggag ggcgtggata tgtcctgcgg gtaaatagct gcgccgatgg 5761 tttctacaaa gatcgttatg tttatcggca ctttgcatcg gccgcgctcc cgattccgga 5821 agtgcttgac attggggaat tcagcgagag cctgacctat tgcatctccc gccgtgcaca 5881 gggtgtcacg ttgcaagacc tgcctgaaac cgaactgccc gctgttctgc agccggtcgc 5941 ggaggccatg gatgcgatcg ctgcggccga tcttagccag acgagcgggt tcggcccatt 6001 cggaccgcaa ggaatcggtc aatacactac atggcgtgat ttcatatgcg cgattgctga 6061 tccccatgtg tatcactggc aaactgtgat ggacgacacc gtcagtgcgt ccgtcgcgca 6121 ggctctcgat gagctgatgc tttgggccga ggactgcccc gaagtccggc acctcgtgca 6181 cgcggatttc ggctccaaca atgtcctgac ggacaatggc cgcataacag cggtcattga 6241 ctggagcgag gcgatgttcg gggattccca atacgaggtc gccaacatct tcttctggag 6301 gccgtggttg gcttgtatgg agcagcagac gcgctacttc gagcggaggc atccggagct 6361 tgcaggatcg ccgcggctcc gggcgtatat gctccgcatt ggtcttgacc aactctatca 6421 gagcttggtt gacggcaatt tcgatgatgc agcttgggcg cagggtcgat gcgacgcaat 6481 cgtccgatcc ggagccggga ctgtcgggcg tacacaaatc gcccgcagaa gcgcggccgt 6541 ctggaccgat ggctgtgtag aagtactcgc cgatagtgga aaccgacgcc ccagcactcg 6601 tccgagggca aaggaatagc acgtgctacg agatttcgat tccaccgccg ccttctatga 6661 aaggttgggc ttcggaatcg ttttccggga cgccggctgg atgatcctcc agcgcgggga 6721 tctcatgctg gagttcttcg cccaccccaa cttgtttatt gcagcttata atggttacaa 6781 ataaagcaat agcatcacaa atttcacaaa taaagcattt ttttcactgc attctagttg 6841 tggtttgtcc aaactcatca atgtatctta tcatgtctgt ataccgtcga cctctagcta 6901 gagcttggcg taatcatggt cattaccaat gcttaatcag tgaggcacct atctcagcga 6961 tctgtctatt tcgttcatcc atagttgcct gactccccgt cgtgtagata actacgatac 7021 gggagggctt accatctggc cccagcgctg cgatgatacc gcgagaacca cgctcaccgg 7081 ctccggattt atcagcaata aaccagccag ccggaagggc cgagcgcaga agtggtcctg 7141 caactttatc cgcctccatc cagtctatta attgttgccg ggaagctaga gtaagtagtt 7201 cgccagttaa tagtttgcgc aacgttgttg ccatcgctac aggcatcgtg gtgtcacgct 7261 cgtcgtttgg tatggcttca ttcagctccg gttcccaacg atcaaggcga gttacatgat 7321 cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt gtcagaagta 7381 agttggccgc agtgttatca ctcatggtta tggcagcact gcataattct cttactgtca 7441 tgccatccgt aagatgcttt tctgtgactg gtgagtactc aaccaagtca ttctgagaat 7501 agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat acgggataat accgcgccac 7561 atagcagaac tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga aaactctcaa 7621 ggatcttacc gctgttgaga tccagttcga tgtaacccac tcgtgcaccc aactgatctt 7681 cagcatcttt tactttcacc agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg 7741 caaaaaaggg aataagggcg acacggaaat gttgaatact catattcttc ctttttcaat 7801 attattgaag catttatcag ggttattgtc tcatgagcgg atacatattt gaatgtattt 7861 agaaaaataa acaaataggg gtcagtgtta caaccaatta accaattctg aacattatcg 7921 cgagcccatt tatacctgaa tatggctcat aacacccctt g //